Kimberly M. Maize

Structural characterization of human histidine triad nucleotide-binding protein 2, a member of the histidine triad superfamily.

The histidine triad proteins (HITs) constitute a large and ubiquitous superfamily of nucleotide hydrolases. The human histidine triad nucleotide‐binding proteins (hHints) are a distinct class of HITs noted for their acyl‐AMP hydrolase and phosphoramidase activity. The first high‐resolution crystal structures of hHint2 with and without bound AMP are described. The differences between hHint2 and previously known HIT family protein structures are discussed. HIT family enzymes have historically been divided into five classes based on their catalytic specificity: Hint, fragile HIT protein, galactose‐1‐phosphate uridylyltransferase, DcpS and aprataxin. However, although several structures exist for the enzymes in these classes, the endogenous substrates of many of these enzymes have not been identified or biochemically characterized. To better understand the structural relationships of the HIT enzymes, a structure‐based phylogeny was constructed that resulted in the identification of several new putative HIT clades with potential acyl‐AMP hydrolase and phosphoramidase activity.

Caught before Released: Structural Mapping of the Reaction Trajectory for the Sofosbuvir Activating Enzyme, Human Histidine Triad Nucleotide Binding Protein 1 (hHint1).

Human histidine triad nucleotide binding protein 1 (hHint1) is classified as an efficient nucleoside phosphoramidase and acyl-adenosine monophosphate hydrolase. Human Hint1 has been shown to be essential for the metabolic activation of nucleotide antiviral pronucleotides (i.e., proTides), such as the FDA approved hepatitis C drug, sofosbuvir. The active site of hHint1 comprises an ensemble of strictly conserved histidines, including nucleophilic His112. To structurally investigate the mechanism of hHint1 catalysis, we have designed and prepared nucleoside thiophosphoramidate substrates that are able to capture the transiently formed nucleotidylated-His112 intermediate (E*) using time-dependent crystallography. Utilizing a catalytically inactive hHint1 His112Asn enzyme variant and wild-type enzyme, the enzyme-substrate (ES1) and product (EP2) complexes were also cocrystallized, respectively, thus providing a structural map of the reaction trajectory. On the basis of these observations and the mechanistic necessity of proton transfers, proton inventory studies were carried out. Although we cannot completely exclude the possibility of more than one proton in flight, the results of these studies were consistent with the transfer of a single proton during the formation of the intermediate. Interestingly, structural analysis revealed that the critical proton transfers required for intermediate formation and hydrolysis may be mediated by a conserved active site water channel. Taken together, our results provide mechanistic insights underpinning histidine nucleophilic catalysis in general and hHint1 catalysis, in particular, thus aiding the design of future proTides and the elucidation of the natural function of the Hint family of enzymes.

Design, Synthesis, and Characterization of Sulfamide and Sulfamate Nucleotidomimetic Inhibitors of hHint1.

Hint1 has recently emerged to be an important target of interest due to its involvement in the regulation of a broad range of CNS functions including opioid signaling, tolerance, neuropathic pain, and nicotine dependence. A series of inhibitors were rationally designed, synthesized, and tested for their inhibitory activity against hHint1 using isothermal titration calorimetry (ITC). The studies resulted in the development of the first small-molecule inhibitors of hHint1 with submicromolar binding affinities. A combination of thermodynamic and high-resolution X-ray crystallographic studies provides an insight into the biomolecular recognition of ligands by hHint1. These novel inhibitors have potential utility as molecular probes to better understand the role and function of hHint1 in the CNS.

Ligand‐induced expansion of the S1′ site in the anthrax toxin lethal factor

The Bacillus anthracis lethal factor (LF) is one component of a tripartite exotoxin partly responsible for persistent anthrax cytotoxicity after initial bacterial infection. Inhibitors of the zinc metalloproteinase have been investigated as potential therapeutic agents, but LF is a challenging target because inhibitors lack sufficient selectivity or possess poor pharmaceutical properties. These structural studies reveal an alternate conformation of the enzyme, induced upon binding of specific inhibitors, that opens a previously unobserved deep pocket termed S1′∗ which might afford new opportunities to design selective inhibitors that target this subsite.

A Crystal Structure Based Guide to the Design of Human Histidine Triad Nucleotide Binding Protein 1 (hHint1) Activated ProTides.

Nucleotide analogues that incorporate a metabolically labile nucleoside phosphoramidate (a ProTide) have found utility as prodrugs. In humans, ProTides can be cleaved by human histidine triad nucleotide binding protein 1 (hHint1) to expose the nucleotide monophosphate. Activation by this route circumvents highly selective nucleoside kinases that limit the use of nucleosides as prodrugs. To better understand the diversity of potential substrates of hHint1, we created and studied a series of phosphoramidate nucleosides. Using a combination of enzyme kinetics, X-ray crystallography, and isothermal titration calorimetry with both wild-type and inactive mutant enzymes, we have been able to explore the energetics of substrate binding and establish a structural basis for catalytic efficiency. Diverse nucleobases are well tolerated, but portions of the ribose are needed to position substrates for catalysis. Beneficial characteristics of the amine leaving group are also revealed. Structural principles revealed by these results may be exploited to tune the rate of substrate hydrolysis to strategically alter the intracellular release of the product nucleoside monophosphate from the ProTide.

Rational Optimization of Mechanism-Based Inhibitors through Determination of the Microscopic Rate Constants of Inactivation

Mechanism-based inhibitors (MBIs) are widely employed in chemistry, biology, and medicine because of their exquisite specificity and sustained duration of inhibition. Optimization of MBIs is complicated because of time-dependent inhibition resulting from multistep inactivation mechanisms. The global kinetic parameters kinact and KI have been used to characterize MBIs, but they provide far less information than is commonly assumed, as shown by derivation and simulation of these parameters. We illustrate an alternative and more rigorous approach for MBI characterization through determination of the individual microscopic rate constants. Kinetic analysis revealed the rate-limiting step of inactivation of the PLP-dependent enzyme BioA by dihydro-(1,4)-pyridone 1. This knowledge was subsequently applied to rationally design a second-generation inhibitor scaffold with a nearly optimal maximum inactivation rate (0.48 min-1).

Errors in Crystal structure of HINT from Helicobacter pylori

The article, Crystal structure of HINT from Helicobacter pylori by Tarique et al. [(2016) Acta Cryst. F72, 42–48], and HINT nomenclature are discussed.

Statistical Analysis, Optimization, and Prioritization of Virtual Screening Parameters for Zinc Enzymes Including the Anthrax Toxin Lethal Factor

The anthrax toxin lethal factor (LF) and matrix metalloproteinase-3 (MMP-3, stromelysin-1) are popular zinc metalloenzyme drug targets, with LF primarily responsible for anthrax-related toxicity and host death, while MMP-3 is involved in cancer- and rheumatic disease-related tissue remodeling. A number of in silico screening techniques, most notably docking and scoring, have proven useful for identifying new potential drug scaffolds targeting LF and MMP-3, as well as for optimizing lead compounds and investigating mechanisms of action. However, virtual screening outcomes can vary significantly depending on the specific docking parameters chosen, and systematic statistical significance analyses are needed to prioritize key parameters for screening small molecules against these zinc systems. In the current work, we present a series of chi-square statistical analyses of virtual screening outcomes for cocrystallized LF and MMP-3 inhibitors docked into their respective targets, evaluated by predicted enzyme-inhibitor dissociation constant and root-mean-square deviation (RMSD) between predicted and experimental bound configurations, and we present a series of preferred parameters for use with these systems in the industry-standard Surflex-Dock screening program, for use by researchers utilizing in silico techniques to discover and optimize new scaffolds.

Identification of Novel Anthrax Toxin Countermeasures Using In Silico Methods

Anthrax is an acute infectious disease caused by the spore-forming, gram-positive, rod-shaped bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF) is the primary anthrax toxin component responsible for cytotoxicity and host death and has been a heavily researched target for design of postexposure therapeutics in the event of a bioterror attack. Various computer-aided drug design methodologies have proven useful for pinpointing new antianthrax drug scaffolds, optimizing existing leads and probes, and elucidating key mechanisms of action. We present a selection of in silico virtual screening protocols incorporating docking and scoring, shape-based searching, and pharmacophore mapping techniques to identify and prioritize small molecules with potential biological activity against LF. We also recommend screening parameters that have been shown to increase the accuracy and reliability of these computational results.

Inhibition by divalent metal ions of human histidine triad nucleotide binding protein1 (hHint1), a regulator of opioid analgesia and neuropathic pain

Human histidine triad nucleotide binding protein 1 (hHint1) is a purine nucleoside phosphoramidase and adenylate hydrolase that has emerged as a potential therapeutic target for the management of pain. However, the molecular mechanism of Hint1 in the signaling pathway has remained less clear. The role of metal ions in regulating postsynaptic transmission is well known, and the active site of hHint1 contains multiple histidines. Here we have investigated the effect of divalent metal ions (Cd2+, Cu2+, Mg2+, Mn2+, Ni2+, and Zn2+) on the structural integrity and catalytic activity of hHint1. With the exception of Mg2+, all the divalent ions inhibited hHint1, the rank of order was found to be Cu2+ >Zn2+ >Cd2+ ≥Ni2+ >Mn2+ based on their IC50 and kin/KI values. A crystal structure of hHint1 with bound Cu2+ is described to explain the competitive reversible inactivation of hHint1 by divalent cations. All the metal ions exhibited time- and concentration- dependent inhibition, with the rate of inactivation highly dependent on alterations of the C-terminus. With the exception of Cu2+; restoration of inhibition was observed for all the metal ions after treatment with EDTA. Our studies reveal a loss in secondary structure and aggregation of hHint1 upon incubation with 10-fold excess of copper. Thus, hHint1 appears to be structurally sensitive to irreversible inactivation by copper, which may be of neurotoxicological and pharmacological significance.